Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that infects over one million people annually and kills over 600,000 individuals per year worldwide. Current treatments are inadequate with high rates of morbidity, mortality, and relapse despite expensive and toxic antifungal interventions. Cryptococcal glycans are crucial determinants of fungal survival and pathogenesis, making them attractive therapeutic targets. The activated donor molecules for synthesis of most glycoconjugates, including the polysaccharide capsule, are nucleotide sugars. These highly charged molecules are typically made in the cytosol and then transported by nucleotide sugar transporters (NSTs) into the secretory pathway, where most glycan biosynthesis occurs. Despite their key role in glycan synthesis, the identity and regulation of the complete set of cryptococcal NSTs remains unknown. This major gap in our knowledge severely limits our ability to manipulate critical biosynthetic processes in this important pathogen. The objective of this proposal is to determine the function of three novel nucleotide sugar transporters, designated NSTX, NSTH, and NSTG, which were identified by homology to known NSTs and have been implicated in fungal virulence. Deletion of these three NSTs markedly reduces capsule synthesis and results in considerable temperature and stress sensitivity. The profound defects in the ability of nstX and nstH mutants to establish infection in mice, furthermore, make these transporters an attractive target for drug discovery. In Aim 1, I will determine the substrate(s) of each of these NSTs using two complementary approaches. I will directly assay transport of radiolabeled nucleotide sugars into the secretory pathway with semi-intact cryptococcal cells. I will also compare glycoconjugate composition of wild-type and mutant cells to determine potential substrates. In Aim 2, I will investigate the mechanism underlying the decreased virulence of the deletion strains. I will examine macrophage phagocytosis and clearance in vitro, and define the dynamics of infection in vivo for each mutant. This work will advance our understanding of glycan biosynthesis and its requirement for virulence. It will thereby set the stage for further studies of fundamental glycobiology, cryptococcal biology and pathogenesis, and potential antifungal agent discovery.